Recent work has convincingly shown that the degree of T cell infiltration plays a critical role in the natural progression of many cancers. A landmak study found that the type, density, and location of cytotoxic T cells within tumors enabled better prediction of patient survival than histopathological methods currently used to stage cancer. However, the tumor microenvironment strongly inhibits expansion and effector functions of tumor-specific T cells. The goal of this project is to identify novel targes for therapy by pinpointing the key genetic and biochemical defects within tumor-infiltrating T cells that restrain their effector function. The identification of such therapeutic targts is of broad relevance in oncology because T cell mediated immune responses have the potential to eradicate cancers. Towards this goal, we propose a novel in vivo shRN discovery approach that enables identification of critical genes and pathways in the relevant microenvironment. Our hypothesis is that shRNAs which target critical inhibitors within dysfunctional T cells can reprogram them to undergo substantial expansion in tumors. T cells will be genetically modified with shRNA pools and then transferred into tumor-bearing mice so that enrichment of particular shRNAs within tumors can be quantified by Illumina sequencing of the shRNA cassette. This in vivo approach will also allow us to address a second related problem in oncology, the identification of combination therapies that act in a highly synergistic manner on defined cellular pathways. We will approach this issue using a lentiviral vector with two shRNA cloning sites, so that an active shRNA can be tested for synergy against a pool of shRNAs. We will determine which shRNA combinations optimize T cell activityin vivo in terms of proliferation, cytokine production and anti-tumor cytotoxic actio. These therapeutic approaches will be tested in a mouse model in which melanomas spontaneously develop based on genetic lesions found in the human disease. A central goal of this effort is to translate these discoveries into clinical application through collaboration with a clinical investigator with expertise in adoptive T cell therapy. Adoptive transfer of T cells that express 'chimeric antigen receptors' (CARs) has emerged as a promising approach because the antibody-like extracellular domain of a CARbinds with high affinity to a surface molecule on tumor cells, while the cytoplasmic domain induces T cell activation. Co-expression of shRNAs and CARs through the same lentiviral vector into T cells could greatly enhance T cell survival and expansion within tumors. This approach will be tested using human T cells in mice bearing human melanomas, as an important step towards clinical translation.